1
The plant specific transcription factors CBP60g and SARD1 are targeted by a
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Verticillium secretory protein VdSCP41 to modulate immunity
3 4
Jun Qin1, Kailun Wang1,2, Lifan Sun1, Haiying Xing1, Sheng Wang1,2, Lin Li3, She
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Chen3, Hui-Shan Guo1,2* and Jie Zhang1,4*
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1
State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese
Academy of Sciences, Beijing, 100101, China. 2
University of Chinese Academy of Sciences, Beijing 100049, China.
3
National Institute of Biological Sciences, Beijing, China.
4
Lead Contact
11 12 13
Corresponding author: Jie Zhang and Hui-Shan Guo
14
Correspondence should be addressed to: Jie Zhang (
[email protected]) or Hui-Shan
15
Guo (
[email protected])
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Tel: 8610-64806355.
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Key Words: Verticillium, effector, calmodulin-binding protein, plant immunity
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ABSTRACT 1
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The vascular pathogen Verticillium dahliae infects the roots of plants to cause
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Verticillium wilt. The molecular mechanisms underlying V. dahliae virulence and host
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resistance remain elusive. Here, we demonstrate that a secretory protein, VdSCP41,
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functions as an intracellular effector that promotes V. dahliae virulence. The
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Arabidopsis master immune regulators CBP60g and SARD1 and cotton GhCBP60b
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are targeted by VdSCP41. VdSCP41 binds the C-terminal portion of CBP60g to
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inhibit its transcription factor activity. Further analyses reveal a transcription
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activation domain within CBP60g that is required for VdSCP41 targeting. Mutations
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in both CBP60g and SARD1 compromise Arabidopsis resistance against V. dahliae
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and partially impair VdSCP41-mediated virulence. Moreover, Virus-induced silencing
33
of GhCBP60b compromises cotton resistance to V. dahliae. This work uncovers a
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virulence strategy in which the V. dahliae secretory protein, VdSCP41, directly targets
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plant transcription factors to inhibit immunity, and reveals CBP60g, SARD1 and
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GhCBP60b as crucial components governing V. dahliae resistance.
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INTRODUCTION The vascular pathogen Verticillium dahliae infects a broad range of plants and 2
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causes devastating diseases. V. dahliae can survive in the form of microsclerotia in
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soil for over ten years (Schnathorst, 1981). During V. dahliae colonization,
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microsclerotia germinate and develop hyphae that adhere tightly to the root surface
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upon the perception of plant roots (Zhao et al., 2014). A few hyphae form hyphopodia
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at the infection site and further differentiate into penetration pegs to penetrate into
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plant cells and colonize the vascular tissue (Fradin and Thomma, 2006; Schnathorst,
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1981; Vallad and Subbarao, 2008; Zhao et al., 2014; Zhao et al., 2016). Although a
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few key steps mediating these infection processes have been elucidated, the molecular
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mechanisms underlying V. dahliae virulence remain largely unknown.
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Progress has been made in isolating virulence factors that are crucial for V.
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dahliae virulence. Several genes that regulate V. dahliae development have been
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characterized as contributing to virulence. VDH1 and VdGARP1, which regulate
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microsclerotial development, are required for V. dahliae virulence in cotton plants
58
(Gao et al., 2010; Klimes and Dobinson, 2006). VdRac1 and VdPKAC1 regulate both
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the development and pathogenicity of the fungus in host plants (Tian et al., 2015;
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Tzima et al., 2010; Tzima et al., 2012). VdEG1, VdEG3, VdCYP1, and VdSNF1 also
61
function as factors that contribute to V. dahliae virulence on cotton (Gui et al., 2017;
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Tzima et al., 2011; Zhang et al., 2016).
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In addition to development-associated virulence factors, fungal pathogens deliver
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effectors that act as virulence factors, inhibiting host defense to promote pathogenesis,
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which also occurs in bacterial and oomycete pathogens. The race 1 strain-specific
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effector Ave1 contributes to V. dahliae virulence on tomato plants not carrying Ve1 (de 3
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Jonge et al., 2012). Necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins
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(NLPs) (NLP1 and NLP2) secreted by V. dahliae strain JR2 are required for its
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pathogenicity in tomato and Arabidopsis plants (Santhanam et al., 2013). VdSge1
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encodes a transcriptional regulator that controls the expression of six putative effector
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genes. Deletion of VdSge1 in V. dahliae results in significantly impaired pathogenicity
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in tomato, suggesting an important role of secreted effectors in suppressing host
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immunity in V. dahliae (Santhanam and Thomma, 2013).
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However, only two secreted effectors of V. dahliae have been indicated to
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function inside the plant cell to modulate host immunity thus far. VdIsc1, a V. dahliae
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effector lacking a known signal peptide, is thought to be delivered into host cells to
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hydrolyze a salicylic acid (SA) precursor and inhibit salicylate metabolism (Liu et al.,
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2014). The small cysteine-containing protein (SCP) VdSCP7 translocates into the
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nucleus of plant cells to either suppress or induce defense in plants through unknown
80
mechanisms (Zhang et al., 2017).
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Plants are equipped with immune components to counteract V. dahliae virulence.
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Tomato Ve1 was identified as an effective resistance locus that recognizes Ave1
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secreted by Race 1 strains (Fradin et al., 2009; Schaible et al., 1951). Genetic analyses
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indicated that EDS1, NDR1 and SERK3/BAK1 are required for Ve1-mediated
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resistance in both tomato and Arabidopsis (Fradin et al., 2011; Fradin et al., 2009).
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Studies in cotton also showed that GhBAK1 and GhNDR1 are crucial components
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regulating defense against V. dahliae (Gao et al., 2013b; Gao et al., 2011),
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demonstrating the common requirements of NDR1 and SERK3/BAK1 in a conserved 4
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defense mechanism against V. dahliae in these plants. GbWRKY1, GhSSN, GbERF,
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GhMLP28, GhMKK2 and GbNRX1 have also been shown to be required for cotton
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resistance against V. dahliae (Gao et al., 2011; Li et al., 2014a; Li et al., 2016; Qin et
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al., 2004; Sun et al., 2014; Yang et al., 2015). A comparative proteomic analysis
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indicated the involvement of both brassinosteroids and jasmonic acid signaling
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pathways in the regulation of cotton resistance to V. dahliae (Gao et al., 2013a).
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Although several regulators have been identified, the mechanisms by which
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plants defend against V. dahliae remain obscure, and further investigation is required
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to isolate more host immune components governing V. dahliae resistance. Targeting
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key immune components is a common strategy employed by pathogenic effectors to
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promote pathogenicity (Boller and He, 2009; Cui et al., 2009; Dou and Zhou, 2012);
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thus, screening of host proteins targeted by pathogenic effectors provides an efficient
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way to identify crucial host defense components.
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The calmodulin-binding proteins (CBPs) functions to bind calmodulin to
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transduce calcium signals (Bouche et al. 2005). The plant-specific CALMODULIN
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BINDING PROTEIN 60 (CBP60) protein family contains eight family members
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including CBP60a–g and SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1
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(SARD1) (Bouche et al. 2005). CBP60g and SARD1 are two most closely related
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members that function partially redundantly in both SA signaling and bacterial
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resistance (Zhang et al. 2010; Wang et al., 2011). CBP60g contains a
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calmodulin-binding domain (CBD) which is essential for its function in defense,
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whereas SARD1 does not bind calmodulin (Wang et al., 2009; Wang et al. 2011; 5
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Zhang et al. 2010). Both CBP60g and SARD1 function as transcription factors that
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directly bind to promoters of genes controlling SA synthesis, such as
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ISOCHORISMATE SYNTHASE 1 (ICS1), ENHANCED DISEASE SUSCEPTIBILITY
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5 (EDS5), NON-EXPRESSOR OF PATHOGENESIS RELATED GENES1 (NPR1)
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(Dong et al., 2004; Nawrath et al., 2002; Sun et al., 2015; Wildermuth et al., 2004).
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Moreover, ChIP-seq analyses have revealed the binding of CBP60g and SARD1 to
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the promoters of a number of genes regulating pathogen-associated molecular patterns
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(PAMPs)-triggered immunity (PTI), effector-triggered immunity (ETI) and systemic
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acquired resistance (SAR) (Sun et al., 2015), indicating their broad role in the
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regulation of plant immunity.In this study, we identified VdSCP41 as a virulence
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effector that suppresses plant immunity induced by PAMPs. VdSCP41 interacts with
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the Arabidopsis CBP60g and SARD1 and modulates their transcription factor activity.
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The contribution of VdSCP41 in V. dahliae virulence is significantly reduced during
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the infection of the cbp60g-1/sard1-1 double mutant. GhCBP60b, the closest protein
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homolog of CBP60g in cotton, is also targeted by VdSCP41 and contributes to cotton
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resistance against V. dahliae. Taken together, our findings revealed that CBP60g,
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SARD1 and GhCBP60b are novel components governing V. dahliae resistance and
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that these proteins are modulated by a secretory effector VdSCP41.
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RESULTS
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VdSCP41 Contributes to V. dahliae Virulence in Arabidopsis and Cotton Plants
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Secretome analyses revealed greater than 700 potential secreted proteins in V. 6
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dahliae (Klosterman et al., 2011). To date, relatively few secreted proteins carrying
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virulence function have been characterized in V. dahliae. We performed a reverse
135
genetic screen to identify secreted proteins that are crucial for V. dahliae virulence.
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Using the newly developed USER-ATMT-DS binary vector (Wang et al., 2016), we
137
constructed 56 gene deletion mutants in V. dahliae strain 592 (V592) that each targets
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an individual potentially secreted protein. The resulting mutants were subjected to
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virulence assessment in host plants, including upland cotton and the model plant
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Arabidopsis. A mutant carrying a targeted deletion of VdSCP41 (VdΔscp41) was
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isolated (Figure 1A) and shown to display significantly reduced virulence compared
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with WT strain V592.
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VdSCP41 encodes a hypothetical protein in the secretome of the V. dahliae Ls.17
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strain (VdLs.17) (Klosterman et al., 2011), and the expression of this gene is
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significantly up-regulated at 2 days post-inoculation (dpi) in Arabidopsis (Figure
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1-figure supplement 1), suggesting a putative function of VdSCP41 in V. dahliae
147
infection. Targeted gene deletion of VdSCP41 in the VdΔscp41 mutant was verified
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by Southern blotting (Figure 1B). The VdΔscp41 mutant displayed much weaker
149
disease symptoms than WT V592 in both upland cotton (Gossypium hirsutum) (Figure
150
1C) and Arabidopsis (Figure 1D). Disease index analyses indicated significantly
151
reduced virulence of the VdΔscp41 mutant compared with that of V592 in both
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upland cotton and Arabidopsis (Figure 1E-F, Figure 1—source data 1). The reduced
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virulence of the VdΔscp41 mutant was restored upon complementation with
7
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GFP-tagged VdSCP41 (VdΔscp41/VdSCP41-GFP) (Figure 1C-F). Thus, VdSCP41
155
functions as a virulence effector that contributes to V. dahliae virulence on host plants.
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VdSCP41 Acts as an Intracellular Effector
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In V. dahliae, some signal peptide-containing SCPs are delivered to the
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septin-organized hyphal neck, which develops from the base of the hyphopodia and
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functions as a fungus-host penetration interface for the dynamic delivery of secretory
161
proteins (Zhou et al., 2017). VdSCP41 contains an N-terminal signal peptide
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predicted by SignalP (http://www.cbs.dtu.dk/services/SignalP/) (Figure 2-figure
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supplement 1A). Therefore, we examined the localization of VdSCP41 in V. dahliae.
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GFP-tagged VdSCP41 (VdSCP41-GFP) was found to localize to the base of the
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hyphopodium and showed ring signals surrounding the hyphal neck when the
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Vd∆scp41
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(Vd∆scp41/VdSCP41-GFP)
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hyphopodium induction (Figure 2A). In contrast, VdSCP41 lacking signal peptide
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(DspVdSCP41-GFP) showed diffused signal at the base of the hyphopodium without
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clear ring signals surrounding the hyphal neck. The results demonstrate that
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VdSCP41-GFP was delivered to the penetration interface for secretion.
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Nuclear
mutant
strain
localization
was
complemented cultured
signal
on
a
(NLS)
with cellophane
sequence
VdSCP41-GFP membrane
for
prediction
173
(http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi) identified a potential
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NLS in VdSCP41 (Figure 2-figure supplement 1A). We therefore took advantage of
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this NLS to examine the putative translocation of V. dahliae-delivered VdSCP41 into 8
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the nucleus of plant cells. The Vd∆scp41/VdSCP41-GFP and GFP-expressing V592
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(V592-GFP) strains were separately inoculated onto onion epidermal cells. While
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GFP fluorescence from the V592-GFP strain was observed in conidial spores,
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VdSCP41-GFP secreted by V. dahliae was capable of translocating into plant cells and
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localizing to the nucleus, in addition to the pericellular space of onion epidermal cells
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(Figure 2-figure supplement 1B). The potential NLS sequence of SCP41 was mutated
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(SCP41-nls-GFP)
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theVdΔscp41/VdSCP41-nls-GFP
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VdSCP41-nls-GFP failed to translocate into the nucleus of onion epidermal cells
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(Figure 2-figure supplement 1B). These results suggested that VdSCP41 delivered by
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V. dahliae translocates into the nucleus of the plant cell, which requires the predicted
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NLS within its sequence.
and
then
complemented strain.
In
into
Vd∆scp41
contrast
to
to
construct
VdSCP41-GFP,
188
We next transiently expressed mCherry-tagged VdSCP41 in plants to further
189
verify its nuclear localization in plant cells. As the signal peptide located at the N
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terminus may guide secreted proteins into the plant extracellular space in some cases,
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we fused mCherry to VdSCP41 both with and without (DspVdSCP41) the signal
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peptide
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DspVdSCP41-mCherry were individually transiently expressed in either Arabidopsis
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protoplasts or Nicotiana benthamiana (N. b.) leaves. mCherry fluorescence imaging
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revealed that both VdSCP41 and DspVdSCP41 localized to the nucleus in
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Arabidopsis cells (Figure 2B). The protein expression level of VdSCP41-mCherry and
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DspVdSCP41-mCherry was detected by immunoblot (Figure 2-figure supplement 1C).
for
analyses
of
subcellular
localization.
9
VdSCP41-mCherry
and
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Similar nuclear localization was observed for both VdSCP41-mCherry and
199
DspVdSCP41-mCherry at the nucleus of N. b. cells (Figure 2-figure supplement 1D).
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These results are consistent with the nuclear localization of the V. dahliae-delivered
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VdSCP41 in onion epidermal cells.
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VdSCP41 Expression in Arabidopsis Inhibits Plant Immunity
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It is believed that the initial function of a fungal effector protein is to suppress
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PTI. To investigate whether VdSCP41 is capable of inhibiting plant immunity when it
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is directly expressed in plants, Arabidopsis transgenic lines expressing DspVdSCP41
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were constructed and assessed for PAMP-induced defense responses. Flg22 is the
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best-characterized PAMP derived from a bacterial pathogen, and it induces the
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expression of PTI -responsive genes in wildtype (WT) Arabidopsis plants. We
210
observed
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VdSCP41-expressing lines (Figure 2-figure supplement 1E), suggesting inhibition of
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PTI conferred by VdSCP41.
reduced
induction
of
flg22-induced
ICS1
in
two
independent
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NLP proteins derived from bacterial, oomycete and fungal organisms have
214
recently been characterized as PAMPs. A conserved 20-amino acid peptide (nlp20)
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within NLP represents the active immunogenic motif that induces PTI in plants
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(Albert et al., 2015; Böhm et al., 2014; Ottmann et al., 2009). We previously reported
217
the immune-inducing activity of VdNLP1 and VdNLP2 derived from V592 in N. b.,
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Arabidopsis, and cotton plants (Zhou et al., 2012). A corresponding peptide located in
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VdNLP2 derived from V592 (nlp20Vd2) was synthesized and shown to cause 10
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up-regulation of cotton PR genes (Du et al., 2017), indicating the immunogenic
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activity of this peptide in cotton. Treatment of Arabidopsis with nlp20Vd2 significantly
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induced ICS1 and FMO1 expression (Figure 2C-D, Figure 2 — source data 1),
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indicating that nlp20Vd2 also exhibits the active immunogenic activity characteristic of
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a PAMP in Arabidopsis. In transgenic lines expressing VdSCP41, but not transgenic
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lines expressing NLS mutated VdSCP41 (DspVdSCP41-nls), suppression of
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nlp20Vd2-induced ICS1 (Figure 2C) and FMO1 (Figure 2D) was observed, suggesting
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that VdSCP41 expression in plants inhibits nlp20Vd2-triggered immunity. ICS1
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encodes the key enzyme controlling SA production and is required for
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pathogen-induced SA accumulation (Wildermuth et al. 2001). We next quantified SA
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production in response to a nonpathogenic bacterial pathogen, Pst DC3000 hrcC-, in
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both WT and DspVdSCP41-expressing plants. In consistent with the suppression of
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PAMP-induced
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accumulated less free SA in response to Pst hrcC- than did WT plants (Figure 2-figure
234
supplement 2A).
ICS1
expression,
transgenic
lines
expressing
DspVdSCP41
235
Inoculation of WT V. dahliae strain V592 on Arabidopsis induced the expression
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of ICS1 and FMO1 (Figure 2-figure supplement 2B-C). This induced expression was
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further enhanced when the plants were inoculated with the VdDscp41 mutant
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compared with V592 (Figure 2-figure supplement 2B-C), indicating that VdSCP41
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suppresses the induced expression of ICS1 and FMO1 during V. dahliae infection.
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Taken together, these results reveal an inhibitory role of VdSCP41 in modulating
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plant immunity. 11
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Arabidopsis Master Immune Regulators CBP60g and SARD1 are Targeted by
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VdSCP41
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Modulating the activity of plant immune components is a common strategy used
246
by effectors to suppress host immunity. To explore the virulence mechanisms
247
employed by VdSCP41 in inhibiting plant immunity, we next searched for plant
248
components that are targeted by VdSCP41. DspVdSCP41 was fused with a 3×FLAG
249
tag and transiently expressed in Arabidopsis protoplasts followed by purification of
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VdSCP41-containing protein complexes. Protein lysates were immuno-precipitated
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using anti-FLAG-conjugated beads and subjected to tandem mass spectrometry. The
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plant CBP CBP60g was identified as a candidate interactor of VdSCP41
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(Supplementary File 1-Table S1).
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CBP60g was then fused with a 3×HA tag and used for reverse
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co-immunoprecipitation (Co-IP) analysis to verify its interaction with VdSCP41.
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FLAG-tagged VdSCP41 was transfected, either alone or together with HA-tagged
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CBP60g, into Arabidopsis protoplasts for transient expression. Anti-HA IP followed
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by an anti-FLAG immunoblot revealed that VdSCP41 was co-purified with CBP60g
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from plant cells (Figure 3-figure supplement 1A). VdSCP41 was further divided into
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an N-terminal portion (VdSCP41N, amino acid 1-213) and a C-terminal portion
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(VdSCP41C, amino acid 163-end),which exhibited an overlap of 50 amino acids, and
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was used to test interactions with CBP60g. Co-IP analysis revealed that VdSCP41C is
263
sufficient for interacting with CBP60g (Figure 3A). 12
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Quantitative luciferase complementation imaging assays were performed to
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further verify the interaction between VdSCP41 and CBP60g in N. b. Co-expression
266
of the N-terminal region of luciferase (NLuc)-tagged BIK1 and the C-terminal region
267
of luciferase (CLuc)-tagged XLG2 driven by the35S promoter was performed as a
268
positive interaction control (Liang et al., 2016). The co-expression of NLuc-VdSCP41
269
and CLuc-CBP60g driven by the 35S promoter in N. b. resulted in much higher
270
luciferase activity than co-expression of NLuc-VdSCP41 and CLuc-XLG2, or
271
CLuc-CBP60g and NLuc-BIK1 (Figure 3B-C, Figure 3—source data 1), confirming
272
the interaction between VdSCP41 and CBP60g in the plants. The expression levels of
273
the NLuc- and CLuc-fusion proteins were further detected via immunoblotting
274
(Figure 3-figure supplement 1B).
275
The nuclear localization of VdSCP41 prompted us to examine whether CBP60g
276
co-localizes with VdSCP41 in the nucleus. GFP-tagged CBP60g mainly localized in
277
the nucleus when it was expressed alone in N. b. cells (Figure 3-figure supplement
278
1C). GFP-tagged CBP60g was co-expressed with mCherry-tagged VdSCP41 without
279
signal peptide (DspVdSCP41-mCherry) in N. b. leaves via Agrobacterium-mediated
280
transient expression. An overlay of the results of GFP and mCherry fluorescence
281
imaging indicated co-localization of DspVdSCP41 and CBP60g in the nucleus in N. b.
282
cells
283
DspVdSCP41-mCherry and DspVdSCP41-nls-mCherry was detected by immunoblot
284
with the indicated antibodies (Figure 3-figure supplement 1D). In addition to
285
co-localization, co-expression of DspVdSCP41-mCherry significantly increased the
(Figure
3D).
The
protein
expression
13
level
of
GFP-CBP60g,
286
nuclear accumulation of CBP60g-GFP (Figure 3D), which was not observed when
287
DspVdSCP41-nls-mCherry with a mutated NLS was co-expressed with CBP60g-GFP
288
(Figure 3D).
289
CBP60g was induced by pathogen and PAMP treatments and required for full
290
resistance against bacterial pathogens (Wang et al., 2011; Zhang et al., 2010b). A
291
closely related protein in the CBP family, SARD1, functions partially redundantly
292
with CBP60g in bacterial resistance (Sun et al., 2015; Wang et al., 2011). We also
293
detected an interaction between VdSCP41 and SARD1 by Co-IP in Arabidopsis
294
protoplasts (Figure 3-figure supplement 2A). VdSCP41 co-localized with SARD1 in
295
N. b. leaves and increased its nuclear accumulation (Figure 3-figure supplement 2B),
296
indicating similar targeting of SARD1 by VdSCP41. It is unlikely an interaction
297
between CBP60g and SARD1 because the luciferase complementation assay did not
298
show interaction between CLuc-tagged CBP60g and NLuc-tagged SARD1 (Figure
299
3-figure supplement 3). Taken together, the above results demonstrated that both
300
CBP60g and SARD1 are targeted by VdSCP41.
301 302
VdSCP41 Interferes with the Transcription Factor Activity of CBP60g
303
CBP60g encodes a plant-specific transcription factor that regulates the
304
expression of a number of defense-related genes. The fact that CBP60g functions as
305
master transcription regulator (Sun et al., 2015; Wang et al., 2011) prompted us to
306
examine whether the targeting of CBP60g by VdSCP41 affects the induction of its
307
target genes. Dual reporter analyses revealed that CBP60g expression in Arabidopsis 14
308
protoplasts significantly enhanced the expression of ICS1::LUC or FMO1::LUC
309
(firefly luciferase) (Figure 4A-B, Figure 4 — source data 1). Co-expression of
310
VdSCP41 inhibited CBP60g-induced ICS1::LUC (Figure 4A) and FMO1::LUC
311
(Figure 4B) expression, whereas the co-expression of VdSCP41N, which is unable to
312
bind CBP60g, was impaired in this inhibition (Figure 4A-B). CBP60g was next
313
divided into an N-terminal portion (CBP60gN, amino acid 1-361) containing its
314
DNA-binding domain and a C-terminal portion (CBP60gC, amino acid 211-end)
315
lacking the functional DNA-binding domain (Zhang et al., 2010b), which exhibited an
316
overlap of 150 amino acids. CBP60gN and CBP60gC were then used to test
317
interactions with VdSCP41. Co-IP analysis showed that VdSCP41 binds to CBP60gC,
318
but not CBP60gN (Figure 4C). The results indicated that VdSCP41 binds the
319
C-terminal portion of CBP60g to interfere with its activity.
320
To test whether VdSCP41 targeting directly affects the DNA-binding activity of
321
CBP60g, recombinant GST-tagged CBP60g and His-tagged VdSCP41C were purified
322
and used for electrophoretic mobility shift assays (EMSAs). GST-CBP60g showed a
323
specific binding to a 60-bp DNA fragment (ICS1 promoter probe) within the ICS1
324
promoter, which is reduced by the addition of unlabelled probe, as previously reported
325
(Zhang et al. 2010) (Figure 4D). The preincubation of VdSCP41C with CBP60g
326
significantly reduced the DNA-binding activity of CBP60g, whereas a soluble
327
fragment of His-tagged VdSCP41 which does not contain its C-terminal portion
328
(VdSCP41100-163) did not (Figure 4D). Another His-tagged V. dahliae protein
329
(VDAG_01962) also did not affect the DNA-binding activity of CBP60g (Figure 4D). 15
330
Coexpression of VdSCP41 did not lead to cleavage or mobility shift of CBP60g
331
(Figure 4-figure supplement 1), suggesting that VdSCP41 is unlikely to act as a
332
protease to target CBP60g. The results proved that binding of VdSCP41C to CBP60g
333
directly inhibits the DNA-binding activity of CBP60g.
334 335
CBP60gC Harbors a Transcription Activation Domain that is Required for
336
Interacting with VdSCP41
337
The binding of VdSCP41 to the C-terminal portion of CBP60g prompted us to
338
test the role of CBP60gC in CBP60g-mediated gene activation. Compared to the full
339
induction of ICS1::LUC or FMO1::LUC by CBP60g (Figure 5A, Figure 5—source
340
data 1), the deletion of the C-terminal portionDC-CBP60g) dramatically
341
compromised its activity to induce both ICS1::LUC and FMO1::LUC (Figure 5A),
342
indicating that CBP60gC is required for CBP60g-mediated gene activation. The
343
results suggest that putative transcription activator activity may be harbored within
344
the CBP60gC. The basic helix-loop-helix (bHLH) transcription factor MYC2 directly
345
binds to the G-box-like (CANNTG) element (Dombrecht et al., 2007; Godoy et al.,
346
2011; Lian et al., 2017) via its bHLH domain to regulate the expression of its target
347
genes, such as the TERPENE SYNTHASE gene 10 (TPS10) (Li et al., 2014b). We
348
therefore took advantage of bHLH-mediated binding to the TPS10 promoter to
349
examine the putative transcription activator activity of CBP60gC. A fragment within
350
the C-terminal portion of CBP60g (amino acid 211-440) activated TPS10::LUC
351
reporter when it was fused with the bHLH domain of MYC2 (bHLH-CBP60g211-440) 16
352
(Figure 5B, Figure 5—source data 1), compared with bHLHMYC2 alone, indicating that
353
the CBP60g211-440 harbors transcription activator activity. Moreover, Co-IP analysis
354
showed that CBP60g211-end, but not CBP60g441-end, co-purified with VdSCP41,
355
indicating the requirement of CBP60g211-440 for binding to VdSCP41 (Figure 5C).
356
Thus, CBP60gC harbors a transcription activation domain, CBP60g211-440, that is
357
required for VdSCP41 targeting.
358
The DC-CBP60g was further co-transfected with CBP60g, together with
359
ICS1::LUC or FMO1::LUC. Dual reporter analyses indicated that co-expression of
360
DC-CBP60g significantly suppressed CBP60g-induced ICS1::LUC and FMO1::LUC
361
(Figure 5A), suggesting a dominant negative effect of DC-CBP60g on the activity of
362
CBP60g.
363 364
CBP60g and SARD1 Contribute to Arabidopsis Resistance against V. dahliae
365
SARD1 functions both redundantly and differentially with CBP60g (Sun et al.,
366
2015; Wang et al., 2011). The finding that VdSCP41 targeted both CBP60g and
367
SARD1 prompted us to assess the roles of CBP60g and SARD1 in resistance to V.
368
dahliae. The WT and cbp60g-1/sard1-1 double mutant plants were inoculated with
369
V592 for the assessment of disease symptoms. As shown in Figure 6, the
370
cbp60g-1/sard1-1 double mutant displayed compromised resistance compared with
371
the WT plants, demonstrating a contribution of CBP60g and SARD1 to V. dahliae
372
resistance.
373
We next investigated the requirement of CBP60g and SARD1 for 17
374
VdSCP41-mediated virulence. The WT and cbp60g-1/sard1-1 double mutant plants
375
were inoculated with the VdDscp41 mutant. The VdDscp41 mutant displayed reduced
376
virulence on the WT plants compared with V592. The reduced virulence arising from
377
VdSCP41 deletion was partially impaired in the cbp60g-1/sard1-1 double mutant
378
plants compared with than in the WT plants (Figure 6, Figure 6—source data 1). The
379
results indicated that both CBP60g and SARD1 are required for full virulence
380
conferred by VdSCP41. However, we still observed reduced virulence of the
381
VdDscp41 mutant compared with V592 on the cbp60g-1/sard1-1 double mutant plants,
382
suggesting the existence of additional targets for VdSCP41 during V. dahliae
383
infection.
384 385
VdSCP41 Interacts with and Co-localizes with GhCBP60b
386
As in Arabidopsis, we observed similar compromised virulence of VdΔscp41
387
mutant in upland cotton (Figure 1E) compared with V592. The results prompted us to
388
test the putative VdSCP41 targeting of GhCBP60b, the closest protein homolog of
389
CBP60g and SARD1 in cotton. Co-IP analysis revealed an interaction between
390
VdSCP41 and GhCBP60b (Figure 7A). To examine the subcellular localization of
391
GhCBP60b, GhCBP60b-GFP was constructed for transient expression in N. b. leaves.
392
GhCBP60b-GFP localized to the nucleus of N. b. cells, and co-expression of
393
GhCBP60b-GFP with VdSCP41-mCherry revealed co-localization of VdSCP41 and
394
GhCBP60b in the nucleus of N. b. cells (Figure 7B), suggesting conserved targeting
395
of Arabidopsis CBP60g and SARD1 and cotton GhCBP60b by VdSCP41.
396 18
397
GhCBP60b is Required for Cotton Resistance against V. dahliae
398
To further examine whether GhCBP60b functions in resistance to V. dahliae in
399
cotton, we generated a virus-induced gene silencing (VIGS) vector (Liu et al., 2002)
400
targeting GhCBP60b (pTRV2-GhCBP60b). Cotton plants were infiltrated with
401
pTRV1 together with pTRV2 or pTRV2-GhCBP60b and further inoculated with V.
402
dahliae. Compared with pTRV2, pTRV2-GhCBP60b-infiltrated cotton plants
403
exhibited higher ratios of wilting (Figure 7C, Figure 7—source data 1) and more
404
severe disease symptoms (Figure 7D-E, Figure 7—source data 1), indicating a role for
405
GhCBP60b in cotton resistance to V. dahliae. The reduced expression of GhCBP60b
406
in pTRV2-GhCBP60b-infiltrated plants was verified via RT-PCR (Figure 7F, Figure 7
407
—source data 1). The results support the targeting of GhCBP60b by VdSCP41 for
408
virulence during V. dahliae infection in cotton.
409 410
DISCUSSION
411
In this study, we identified VdSCP41 as an intracellular effector that is crucial
412
for V. dahliae virulence. VdSCP41 targets Arabidopsis CBP60g and SARD1 to
413
interfere with the induction of their target genes. CBP60g and SARD1 are required for
414
Arabidopsis resistance to V. dahliae and VdSCP41-mediated virulence. Silencing of
415
GhCBP60b compromised cotton resistance to V. dahliae. The results revealed a
416
conserved and important role for Arabidopsis CBP60g and SARD1 and cotton
417
GhCBP60b, which are modulated by an intracellular effector, in plant resistance
418
against V. dahliae. 19
419 420
VdSCP41 is Secreted by V. dahliae and Translocates into Plant Cells
421
To actively suppress plant defense or modulate host physiology to benefit
422
pathogenic fitness, oomycete and fungal pathogens deliver hundreds of effectors
423
through specialized intracellular fungal structures, such as haustoria and infection
424
hyphaea, into the host apoplastic space or directly into plant cells (Lo Presti et al.,
425
2015). Although haustoria have not been observed during infection, V. dahliae
426
develops a penetration peg from a hyphopodium when infecting plant roots (Zhao et
427
al., 2016). During V. dahliae infection, the penetration peg developed from the
428
hyphopodium further develops a hyphal neck and forms a septin ring that partitions
429
the hyphopodium and invasive hyphae. This septin-organized apparatus functions as a
430
fungus-host interface for the dynamic delivery of secretory proteins, such as SCPs
431
(Zhao et al., 2016; Zhou et al., 2017).
432
To date, relatively few V. dahliae-secreted effectors have been characterized as
433
functioning inside host cells to modulate host immunity. Here, we provided evidence
434
of the secretion and translocation of VdSCP41 secreted by V. dahliae into plant cells.
435
VdSCP41 contains a signal peptide and localizes to the base of the hyphopodium to
436
form a septin-like ring during infection (Figure 2A), indicating that it is secreted via
437
the septin-organized apparatus at the fungus-host interface. The localization of
438
VdSCP41 delivered by V. dahliae at the nucleus of onion epidermal cells (Figure
439
2-figure supplement 1B) suggested the translocation of VdSCP41 into plant cells. A
440
few conserved motifs have been indicated to serve as signals for the uptake of fungal 20
441
or oomycete effectors into plant cells. For example, a few effectors secreted by cereal
442
powdery mildew and rust pathogens possess a Y/F/WxC motif that serves as a signal
443
for translocation into the plant cell (Godfrey et al., 2010; Spanu et al., 2010). A set of
444
oomycete effectors contain a RXLR-dEER motif that appears to assist in targeting
445
effectors into plant cells (Dou et al., 2008; Whisson et al., 2007). Another set of
446
oomycete effectors possess a FLAK motif for translocation (Schornack et al., 2010).
447
However, VdSCP41 lacks any of the above conserved motifs, and the mechanisms by
448
which assist the uptake of VdSCP41 into plant cells remain unclear.
449
VdSCP41 Targets CBP60g and SARD1 and Interferes with Plant Defense
450
CBP60g and SARD1 are master regulators in immunity. ChIP-seq analyses
451
allowed the identification of a large number of targets genes for CBP60g and SARD1
452
and support a model in which CBP60g and SARD1 accumulate in the plant nucleus
453
and act as master regulators, to activate both positive and negative immune regulators
454
(Sun et al., 2015; Wang et al., 2009; Zhang et al., 2010b). Genetic evidence revealed
455
that CBP60g and SARD1 are positive immune regulators required for immune
456
responses and bacterial resistance (Wang et al., 2009; Zhang et al., 2010b). We
457
demonstrated that VdSCP41 targets CBP60g and SARD1 and interferes with their
458
activity, thus supporting the virulence function of VdSCP41 for immune suppression.
459
Consistent with the inhibition of CBP60g and SARD1 activity, we observed less
460
pathogen-induced SA accumulation in transgenic lines expressing VdSCP41 than in
461
WT plants (Figure 2-figure supplement 2A). As CBP60g and SARD1 also directly
462
bind to the promoters of both ALD1 and SARD4 (Sun et al., 2018), two major genes 21
463
encoding pipecolic acid (Pip) biosynthesis enzymes, to regulate Pip biosynthesis, a
464
potential contribution of Pip to V. dahliae resistance will be of interest for further
465
investigation.
466
We showed that VdSCP41 binds the C-terminal portion of CBP60g to interfere
467
with its transcription factor activity (Figure 4A-B). Coexpression of VdSCP41 did not
468
lead to cleavage or mobility shift of CBP60g (Figure 4-figure supplement 1),
469
suggesting that VdSCP41 is unlikely to act as a protease to target CBP60g. In addition,
470
CBP60g211-440, a domain within the C-terminal portion of CBP60g, harbors
471
transcription activator activity (Figure 5B) and is required for interacting with
472
VdSCP41 (Figure 5C). It is likely that CBP60gC functions to promote transcriptional
473
activation via recruiting additional activators, and binding of VdSCP41 interrupts the
474
activity of this domain or the recruitment of associated activators via this domain. The
475
dominant negative function of DC-CBP60g (Figure 5A) supports a dominant negative
476
effect on CBP60g activity arising from increased nuclear accumulation of
477
VdSCP41-impaired CBP60g. Thus, VdSCP41 mediated over-accumulation of
478
CBP60g provides an additional strategy to further interfere with its transcription
479
factor activity. The results suggest a novel virulence strategy in which a pathogenic
480
effector directly targets host transcription factors to interfere with their activity and
481
modulate plant immunity. However, it is equally possible that the increased nuclear
482
accumulation of CBP60g results from the feed-back regulation as a result of impaired
483
CBP60g function.
484
VdSCP41 Functions to Suppress Immunity 22
485
CBP60g and SARD1 are key components of the SA signaling pathway, which
486
serves as an attractive target for bacterial and fungal effectors (DebRoy et al., 2004;
487
Djamei et al., 2011; Nomura et al., 2011). Targeting of CBP60g and SARD1 by
488
VdSCP41 may therefore interfere with plant resistance to V. dahliae via the
489
manipulation of SA signaling. On the other hand, ChIP-seq analyses identified a
490
number of SA-independent regulators that are directly targeted by CBP60g and
491
SARD1 (Sun et al., 2015), revealing broader and SA-independent functions of
492
CBP60g and SARD1 in immunity. It is likely that signaling pathways other than SA
493
are also targeted by VdSCP41 through CBP60g and SARD1 to interfere with plant
494
immunity. Consistent with this assumption, we showed that nlp20Vd2 functions to
495
trigger the induction of both SA-dependent and SA-independent defense genes during
496
V. dahliae infection (Figure 2C-D). Furthermore, VdSCP41 expression suppressed the
497
induction of both SA-dependent ICS1 and SA-independent FMO1 by nlp20Vd2 (Figure
498
2C-D). Modulation of CBP60g and SARD1 may result in interference with their
499
function as master transcription factors in PTI and in other defense pathways.
500
Taken together, our results support a model in which PAMPs from V. dahliae,
501
such as NLPs and chitins, are recognized by plants to induce the expression of
502
CBP60g and SARD1, which subsequently regulate the expression of a number of
503
immune regulators to defend against pathogen infection (Figure 8A). VdSCP41
504
secreted by V. dahliae functions as an intracellular effector that targets the
505
transcription activator domain of CBPs, interrupting the function of this domain, to
506
interfere with their transcription factor activity, and thus modulates both 23
507
SA-dependent and SA-independent regulators to inhibit plant immunity against V.
508
dahliae (Figure 8B).
509
24
510
MATERIAL AND METHODS Key Resources Table Reagent type (species) or
Designation
resource gene (Verticillium dahliae)
VdSCP41
gene (Gossypium hirsutum)
GhCBP60b
strain, strain background (Verticillium dahliae) other (cotton plant) antibody antibody antibody antibody
Source or reference PMID:21829347
PMID:21151869
Xinluzao No. 16
PMID:28282450
monoclonal) anti-HA (mouse monoclonal) anti-GFP (mouse monoclonal) anti-CLuc (mouse monoclonal)
Additional information
NCBI Gene ID:20709665 NCBI Gene ID:107899061
V592
anti-FALG (mouse
Identifiers
Sigma
RRID:AB_259529
1:10000 dilution
Roche
RRID:AB_514506
1:5000 dilution
Roche
RRID:AB_390913
1:3000 dilution
Sigma
RRID:AB_439707
1:3000 dilution
antibody
anti-mCherry
Easybio
1:3000 dilution
peptide
nlp20Vd2
PMID:28755291
recombinant DNA reagent
pTRV1 (VIGS vector)
PMID:12220268
recombinant DNA reagent
pTRV2 (VIGS vector)
PMID:12220268
511 25
512
Fungal strains, plant materials and antibodies.
513
The Verticillium dahliae strain V592 (Gao et al., 2010) was used in this study.
514
Verticillium dahliae strains were grown on potato dextrose agar (PDA) medium at
515
25°C in the dark. To collect conidia, the mycelial plugs were cultured in potato
516
dextrose broth (PDB) liquid medium with shaking at 200 rpm at 25°C for 3-5 days.
517
Cotton plants (`Xinluzao No. 16') were used for virulence assessment in this study
518
(Zhou et al., 2017). Arabidopsis thaliana plants used in this study includes Col-0
519
(wild-type), the cbp60g-1/sard1-1 mutant (Zhang et al., 2010b). VdSCP41 was
520
amplified from the V592 cDNA and cloned into pCambia1300-35S-FLAG (Zhang et
521
al., 2010a) to construct Arabidopsis transgenic lines expressing VdSCP41. Antibodies
522
used
523
(RRID:AB_514506), anti-CLuc (RRID:AB_439707), anti-GFP (RRID:AB_390913),
524
and anti-mCherry (Easybio, BE2026).
525
Generation of deletion and complementation fungal strains
526
The upstream and downstream flanking sequences were PCR amplified from V592
527
genomic DNA and cloned into pGKO-HPT vector (Wang et al., 2016). The resulting
528
construct was transformed into Agrobacterium tumefaciens EHA105, and used for
529
Agrobacterium tumefaciens-mediated transformation (ATMT) to generate the
530
VdΔscp41 mutant strain (Wang et al., 2016). Genomic region, including 1.5 kb
531
upstream from the start codon, of VdSCP41 was amplified and cloned into
532
pNat-Tef-TrpC vector (Zhou et al., 2017) to generate construct for complementation.
533
The resulting construct was transformed into Agrobacterium tumefaciens EHA105,
in
this
study
includes
anti-FLAG
26
(RRID:AB_259529),
anti-HA
534
and used for ATMT to generate VdΔscp41/VdSCP41 strain. Primers used in this
535
study are listed in Supplementary File 2.
536
Protein complex purification and mass spectrum analysis
537
Arabidopsis protoplasts isolated from 10 gram leaves were transfected with
538
DspVdSCP41-FLAG, DspVdSCP45-FLAG, or empty vector for protein expression.
539
Transfected protoplasts were collected and total protein was extracted with extraction
540
buffer containing 50mM HEPES, pH7.5, 150 mM KCl, 1 mM EDTA, 1 mM DTT,
541
0.2% Triton X-100, and 1×proteinase inhibitor cocktail. Total protein was incubated
542
with 50μl anti-FLAG agarose beads (sigma) for 12 hours at 4°C. The
543
immunocomplex was washed three times using the above buffer and eluted with
544
100μl 1μg/μl 3×FLAG peptide. The eluted proteins were run 10 mm into the
545
separating gel and stained with Proteo Silver stain kit (Sigma). Total protein was
546
destained and digested in-gel with sequencing grade trypsin (10 ng/mL trypsin, 50
547
mM ammonium bicarbonate [pH 8.0]) overnight. Peptides were sequentially extracted
548
with 5% formic acid/50% acetonitrile and 0.1% formic acid/75% acetonitrile and
549
concentrated to 20 ml. The extracted peptides were separated by an analytical capillary
550
column packed with 5 mm spherical C18 reversed-phase material. The eluted peptides
551
were sprayed into a LTQ mass spectrometer (Thermo Fisher Scientific) equipped with
552
a nano-ESI ion source. The mass spectrometer was operated in data-dependent mode
553
with one MS scan followed by five MS/MS scans for each cycle. Database searches
554
were performed on an in-house Mascot server (Matrix Science Ltd., London, UK)
555
against IPI (International Protein Index) Arabidopsis protein database. 27
556
Co-immunoprecipitation assay in protoplasts
557
Five-week-old
558
pUC-35S-VdSCP41-FLAG, or its variants construct, was co-transfected with
559
pUC-35S-CBP60g-HA,
560
pUC-35S-GhCBP60b-HA, into Arabidopsis protoplasts. Total protein was extracted
561
with extraction buffer. For anti-HA IP, total protein was incubated with 2 μg of
562
anti-HA antibody together with protein A agarose at 4°C for 4 hours. The agarose
563
were collected and boiled for 5 minutes with 1× protein loading buffer.
564
Immunoprecipitates were separated by 10% SDS-PAGE and the presence of
565
VdSCP41-FLAG or its variants, CBP60g-HA or its variants， SARD1-HA, or
566
GhCBP60b-HA was detected by anti-FLAG or anti-HA immunoblot respectively.
567
SA Measurement
568
SA was extracted and measured following the method described previously (Sun et al.,
569
2015). Around 0.3 gram leaf tissue collected from 4-week-old plants was ground into
570
powder in liquid nitrogen. Plant leaves were infiltrated with or without Pst DC3000
571
hrcC- (OD600=0.1) 12 hours before sample collection. Three samples for each
572
treatment were analysed. The samples were extracted with 0.8 mL 90% methanol and
573
sonicated for 15 min, and the supernatant was transferred into a new tube. The pellet
574
was re-extracted with 0.5 mL of 100% methanol, the supernatant was combined with
575
the first-step supernatant and dried by vacuum. The pellet was resuspended in 500 μL
576
0.1 M sodium acetate (pH 5.5) in 10% methanol. An equal volume of 10% TCA was
577
added and the samples were vortexed, sonicated for 5min. After centrifuged, the
Arabidopsis
or
plants
its
were
variants
28
used
construct,
for
protoplast
isolation.
pUC-35S-SARD1-HA,
or
578
supernatant was extracted three times with 0.5 ml of extraction buffer
579
(ethylacetate/cyclopentane/isopropanol:100/99/1 by volume). After spinning, the
580
organic phases were collected and dried by vacuum. The samples were then dissolved
581
in 250 μL 100% methanol and filtered through a 0.22 μm filter. The samples were
582
then assayed by HPLC-MS/MS analysis on AB SCIEX QTRAP 4500 system (AB
583
SCIEX, Foster, CA, USA).
584
Luciferase complementation imaging assay
585
Agrobacterium tumefaciens GV3101 strain carrying CLuc or NLuc tagged consctruct
586
were infiltrated into leaves of 4-week-old Nicotiana benthamiana. LUC activity in
587
leaves was examined 2 days post infiltration. N. b. leaves were treated with 1 mM
588
luciferin and kept in dark for 5 min to quench the fluorescence. LUC image was
589
captured by CCD imaging apparatus and the quantitative LUC activity was
590
determined by microplate luminometer. Expression of CLuc-tagged proteins or
591
NLuc-tagged proteins was detected by anti-CLuc or anti-HA western blot,
592
respectively.
593
Electrophoretic mobility shift assays (EMSAs)
594
The full-length CBP60g was cloned into the pGEX-6p-1 vector. VdSCP41C,
595
VdSCP41100-163 or VDAG_01962 was cloned into the pET-28a vector. The resulting
596
constructs were transformed into in E. coli BL21 (DE3) competent cells. BL21 (DE3)
597
strains containing the expression vectors were cultured and induced by IPTG at 16°C
598
for 16 hours. GST-tagged full-length CBP60g, His-tagged VdSCP41C or
599
VdSCP41100-163, and His-tagged VDAG_01962 recombinant protein were affinity 29
600
purified and used for EMSAs. A 60-bp probe within the DNA fragment 7 (Zhang et
601
al., 2010) in the ICS1 promoter were labeled with [γ-32P]ATP using T4 polynucleotide
602
kinase. Binding reactions were carried out in a 20μl volume of reaction buffer [10
603
mM Tris-HCl, pH 7.5, 50 mM KCl, 1mM DTT, 1 μl 50 ng/μl poly(dI-dC)] for 30 min
604
at room temperature. Labeled DNA probe (2 fmol) was incubated with 4 μg
605
GST-CBP60g. 150× unlabeled DNA probe was used for competition. 1.5 μg
606
His-tagged VdSCP41C163-end, VdSCP41100-163, or VDAG_01962 was preincubated
607
with GST-CBP60g for 30 min at room temperature before DNA binding. The reaction
608
was stopped by adding DNA loading buffer and the samples were separated by a 5%
609
native PAGE gel. After electrophoresis, the gel was autoradiographed.
610
RNA isolation and qRT-PCR
611
Total RNA was isolated with the TRIzol reagent (Invitrogen) and used for cDNA
612
synthesis with SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen)
613
following the instructions provided by the manufacturer. The quantitative PCR were
614
performed with SYBR Premix Ex Taq kit (TaKaRa) following standard protocols.
615
Arabidopsis col-0 or transgenic plants were treated with H2O, 1μM flg22 or nlp20Vd2
616
(Du et al., 2017) as indicated for 3 hours. RNA was isolated and used for RT-PCR
617
analysis for the expression of ICS1 and FMO1. AtTUB4 was used as internal control.
618
To detect gene expression in cotton plants, leaves from cotton plants were collected
619
14 days post Agrobacterium infiltration. Quantitative RT-PCR was performed as
620
described above. G. hirsutum HISTONE3 was used as internal control. For RT-PCR
621
analyses of VdSCP41, V592 or VdΔscp41 strain was incubated with the roots of 30
622
7-day-old Arabidopsis col-0 plants for 2 days. Conidia were collected for RNA
623
isolation and RT-PCR analysis for the expression of VdSCP41. For RT-PCR analyses
624
of V. dahliae-infected Arabidopsis plants, Arabidopsis plants infected with or without
625
V592 or VdΔscp41 strain were collected for RNA isolation and RT-PCR analysis for
626
the expression of ICS1 and FMO1. VdELF1 was used as internal control for VdSCP41.
627
AtTUB4 was used as internal control for ICS1 and FMO1.
628
Fluorescence microscopy
629
Agrobacterium tumefaciens EHA105 strain carrying pCambia1300-35S-CBP60g-GFP,
630
pCambia1300-35S-SARD1-GFP,
631
infiltrated alone, or together with Agrobacterium tumefaciens EHA105 strain carrying
632
pCambia1300-35S-VdSCP41-mCherry (or its variants mutants) into leaves of
633
4-week-old N. b. GFP and mCherry fluorescence were observed with Leica SP8
634
confocal microscopy 3 days post infiltration. The intensity of fluorescent signals was
635
determined by Image J software. For fluorescence microscopy in Arabidopsis,
636
Arabidopsis protoplasts were transfected with 35S-CBP60g-GFP alone, or together
637
with 35S-VdSCP41-mCherry (or its variants mutants). The protoplasts were incubated
638
overnight under faint light before GFP and mCherry fluorescence were observed. To
639
examine the subcellular localization of VdSCP41, conidia were cultured on
640
cellophane and incubated for 3-9 days before microscopy observation. The cellophane
641
with mycelium were collected and observed as described (Zhou et al., 2017). The
642
plasma membrane of the fungi was stained with FM4-64 (red).
643
Reporter Assay in Arabidopsis Protoplast
or
pCambia1300-35S-GhCBP60b-GFP
31
was
644
Arabidopsis protoplasts were co-transfected with ICS1::LUC or FMO1::LUC, and
645
35S::RLUC (Renilla luciferase), or together with VdSCP41, CBP60g, or their variants
646
as indicated. 12 hours after transfection, protein of transfected protoplasts was
647
isolated, and LUC activity was determined by using Dual-Luciferase® Reporter
648
system (Promega) following manufacture’s instruction.
649
Virus-induced gene silencing in cotton plants
650
The VIGS was performed as described (Gao and Shan, 2013). Cotton plants were
651
grown at 23-25°C in the growth room until two cotyledons have emerged. A 465 bp
652
fragment of GhCBP60b cDNA was PCR amplified from G. hirsutum and cloned into
653
pTRV2 plasmid (Liu et al., 2002). Agrobacterium strain carrying pTRV1 together
654
with Agrobacterium strain carrying pTRV2 or pTRV2-GhCBP60b, as indicated, was
655
infiltrated into the cotyledons of the cotton plants. The cotton plants were root-dip
656
inoculated by V. dahliae V592 2 weeks post Agrobacterium infiltration.
657
Infection assay
658
Cotton or Arabidopsis plants were infected by the root-dip inoculation method (Gao et
659
al., 2010). A conidial suspension of 107/ml from the indicated stains was used as the
660
inoculums. The disease grade was classified as follows: Grade 0 (no symptoms), 1
661
(0-25% wilted leaves), 2 (25-50%), 3 (50-75%) and 4 (75-100%). The disease index
662
was calculated as 100 × [sum (number of plants × disease grade)] / [(total number of
663
plants) × (maximal disease grade)] (Xu et al., 2014). The onion epidermis infection
664
assay was performed as described (Zhang et al., 2017). A conidial suspension of
665
107/ml from V592-GFP, VdΔscp41/SCP41-GFP or VdΔscp41/SCP41-nls-GFP strain 32
666
was inoculated onto the inner layer of onion epidermal cells and incubated on 1%
667
water agar plates for 3-5 days before confocal imaging.
668 669
ACKNOWLEDGMENTS
670
The authors are grateful to Prof. Yuelin Zhang for providing Arabidopsis
671
cbp60g-1/sard1-1 mutant, to Prof. Yule Liu for providing the pTRV1 and pTRV2
672
plasmids, to Prof. Jian Ye for providing the TPS10::LUC plasmid, to Yao Wu for help
673
in SA measurement. This work was supported by grants from the Strategic Priority
674
Research Program of the Chinese Academy of Sciences (XDB11020600) to J. Z., the
675
National Natural Science Foundation of China (31730078) to H-S.G., the Strategic
676
Priority Research Program of the Chinese Academy of Sciences (XDB11040500) to
677
H-S.G., the Youth Innovation Promotion Association of the Chinese Academy of
678
Sciences, and the National Natural Science Foundation of China (31571968,
679
31501593), and the State Key Laboratory of Plant Genomics, Chinese Academy of
680
Sciences.
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Hyphopodium-specific
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Figure Legends
901
Figure 1. VdSCP41 contributes to V. dahliae virulence on host plants.
902
(A) Schematic description of the generation of the VdΔscp41 mutant. (B) Southern
903
blot analysis of the VdSCP41 gene deletion in the VdΔscp41 mutant. Genomic DNA
904
samples isolated from V592 and VdΔscp41 strains were digested by BamHI and
905
subjected to Southern blot analysis. (C-D) Disease symptoms of upland cotton (C)
906
and Arabidopsis (D) plants infected with the wildtype (V592), VdΔscp41, and
907
VdΔscp41/VdSCP41-GFP strains, as indicated. (E-F) Disease index analyses of 43
908
upland cotton (E) and Arabidopsis (F) infected with the indicated strains. The plants
909
were photographed and subjected to disease index analyses 3-4 weeks post
910
inoculationThe disease indexes were evaluated with three replicates generated from
911
24 plants for each inoculum. Error bars indicate standard deviation of three biological
912
replicates. Student’s t test was carried out to determine the significance of difference.
913
* indicates significant difference at P value of
